This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of the polynucleotides and polypeptides; processes for making the polynucleotides and the polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of the polynucleotides, polypeptides, variants, derivatives, agonists and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of human cytostatin II.
The growth and differentiation of cells and the development of tissues and glands is controlled by autocrine and paracrine factors, such as systemic hormones and factors that modulate or mediate the action of hormones, such as growth factors, which themselves may be hormones.
For example, peptides that locally signal growth cessation and stimulate differentiation of cells of the developing epithelium are very important to mammary gland development. These factors largely have not been identified or characterized, particularly not in humans.
A few factors that play a role in the humoral mediation of growth and differentiation of cells in tissues and glands, mammary glands in particular, have been identified in non-human organisms. One such factor is mammary-derived growth inhibitor (xe2x80x9cMDGIxe2x80x9d), which, at least in mice and cows, inhibits epithelial cell growth and stimulates epithelial cell differentiation. MDGI was first identified in milk and mammary glands of cows. Subsequently, it was identified in mice.
MDGI occurs in at least two forms produced by alternative routes of post-translational processing. The original form is referred to as MDGI and the second form is called MDGI-2.
MDGI is associated primarily with milk fat globule membranes (xe2x80x9cMFGMxe2x80x9d), as assessed by immunological assays using anti-MDGI antibodies. Similar time course studies show that MDGI increases dramatically in mammary glands when lactation begins, following delivery. MDGI-2 differs from MDGI in this respect. It is found in mammary glands during pregnancy but not during lactation.
The roles of the two forms of MDGI and their mechanism(s) of action are not clearly defined. Mouse and bovine MDGI are homologous to one another and to a family of low molecular mass hydrophobic ligand-binding proteins (xe2x80x9clow MW HLBP(s)xe2x80x9d), which includes fatty acid-binding proteins (xe2x80x9cFABP(s)xe2x80x9d) from brain, heart, liver and intestine, myelin P2 protein, the differentiation associated protein of adipocytes called p422 gastrotropin and cellular retinoic acid-binding protein (xe2x80x9cCRABPxe2x80x9d). These proteins, which bind hydrophobic ligands such as long-chain fatty acids, retinoids and eiconsanoids, are thought to play roles in the transport, sequestration, or metabolism of fatty acids and fatty acid derivatives. However, they are expressed in a differentiation specific manner, in cells of the mammary gland, heart, liver, brain and intestine, and they appear not only to play roles in basal metabolism but also to have important roles in differentiation and development.
The homology of MDGI to the low MW HLBPs raises the possibility that MDGI, at least as part of its function, binds a hydrophobic ligand, and that binding to this ligand is important to the mechanisms by which MDGI inhibits cell growth and stimulates differentiation; although all the other low MW HLBPs except gastrotropin act intracellularly, whereas MDGI acts extracellularly, in vitro.
Among the low MW HLBPs, MDGI most closely resembles the fatty acid binding proteins (xe2x80x9cFABPxe2x80x9d). FABPs have been identified in brain, heart, liver and intestine. Heart FABP, like MDGI, whether produced from natural sources or by expression of a cloned gene in a heterologous host, inhibits growth of normal mammary epithelial cells (xe2x80x9cMECxe2x80x9d) of mouse origin. In addition, it stimulates milk protein synthesis and it stimulates its own expression in these cells. However, unlike bovine heart FABP, bovine MDGI does not bind fatty acids, although the two proteins are 95% homologous and it has been suggested that heart FABP actually may be a form of MDGI. Thus, even if MDGI is a low MW HLBP, its substrate affinities are distinct from its close relatives in the family, and it therefore likely plays a different physiological role.
In vivo MDGI is found in capillary endothelial cells and in the mammary parenchyma, in mice and cows. MDGI appears first in the capillary endothelial cells and later in the secretory epithelial cells. The location of MDGI in the mammary capillary endothelium is consistent with a role in regulating endothelial cell proliferation.
A number of activities of MDGI have been demonstrated in vitro. For instance, it has been shown that MDGI inhibits L(+)-lactate-, arachidonic acid- and 15-S-hydroxyeicosatetraenoic acid-induced supersensitivity of neonatal rat heart cells to beta-adrenergic stimulation. The induced hypersensitivity is mediated by a small population of beta 2-adrenergic receptors and, therefore, it has been suggested that MDGI interferes with the normal function of these receptors. Interaction with these receptors might also be part of the mechanism by which MDGI inhibits cells growth. This activity also raises the possibility that MDGI naturally modulates the beta-adrenergic sensitivity of cardiac myocytes
The effect of MDGI on differentiation of mammary epithelial cells (xe2x80x9cMECxe2x80x9d) has been further demonstrated by antisense inhibition experiments using phosphorothioate oligonucleotides. These experiments show that MDGI antisense molecules decrease beta-casein levels and suppress the appearance of alveolar end buds in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of epidermal growth factor, and epidermal growth factor antagonizes the activities of MDGI. MDGI is the first known growth inhibitor which promotes mammary gland differentiation.
The regulatory properties of MDGI can be fully mimicked by an 11-amino acid sequence, which is represented in the carboxyl terminus of MDGI and a subfamily of the low MW HLBPs.
Not all mammary epithelial cell lines respond to MDGI in the same way. MDGI inhibits growth of normal human MEC, passaged for varying lengths of time. It also inhibits growth of the mouse mammary malignant epithelial cell lines mMaCa 20177, the human malignant mammary cell lines MaTu and T47D and it inhibits the resumption of growth of stationary Ehrlich ascites carcinoma cells (xe2x80x9cEAC xe2x80x9d) in vitro. In contrast, MDGI slightly stimulates growth of the human malignant mammary epithelial cell line MCF7. Finally, MDGI promotes differentiation of mouse pluripotent embryonic stem cells.
The mechanism of the effects of MDGI on cells is not known, as yet. The resumption of growth of stationary Ehrlich ascites carcinoma cells (xe2x80x9cEACxe2x80x9d) in vitro is accompanied by a rapid increase in cellular c-fos, c-myc and c-ras mRNA. The rapid induction of these genes upon exposure to MDGI underscores the importance of oncogene expression to growth regulation and evidences a positive correlation between cell growth and expression of c-fos, c-myc and c-ras. Furthermore, the effect of MDGI on expression of these genes indicates that it is a positive effector of cellular protooncogene expression, either directly or through one or more signaling pathways, or both.
It also has been shown that MDGI can function as a potent tumor suppressor gene. Human breast cancer cells transfected with a MDGI expression construct exhibited differentiated morphology, reduced proliferation rate, reduced clonogenicity in soft agar, and reduced tumorgenicity in nude mice. The human homologue of this gene was mapped to chromosome 1p33-35, a locus previously shown to exhibit frequent loss of heterozygosity in human breast cancer (about 40% of tumors). The magnitude of the in vivo and in vitro tumor suppressor activity of MDGI is comparable to that previously observed for BRCA1, p53, Rb, and H19.
The effects of MDGI on cell growth and differentiation, and on expression of cellular protooncogene expression reiterate the importance of soluble factors in normal growth and differentiation of cells, tissues, glands and organs, and their roles in aberrant cell growth, dysfunction and disease. Clearly, there is a need for factors that regulate growth and differentiation of normal and abnormal cells. There is a need, therefore, for identification and characterization of such factors that modulate growth and differentiation of cells, both normally and in disease states. In particular, there is a need to isolate and characterize additional cytostatins that modulate growth and differentiation of cells such as epithelial cells, particularly mammary epithelial cells, that are essential to the proper development and health of tissue and organs such as mammary glands of developing and adult human females.
Toward these ends, and others, it is an object of the present invention to provide polypeptides, inter alia, that have been identified as novel cytostatins by homology to known cytostatins, such as MDGI, of the amino acid sequence set out in FIG. 1 (SEQ ID NO:2).
It is a further object of the invention, moreover, to provide polynucleotides that encode cytostatins, particularly polynucleotides that encode the polypeptide herein designated cytostatin II.
In a particularly preferred embodiment of this aspect of the invention the polynucleotide comprises the region encoding human cytostatin II in the sequence set out in FIG. 1 (SEQ ID NO:1) or in the cDNA in ATCC deposit No. 97287 (referred to herein as the deposited clone).
In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding human cytostatin II, including mRNAs, DNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives.
Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of human cytostatin II.
It also is an object of the invention to provide cytostatin II polypeptides, particularly human cytostatin II polypeptides, that modulate growth activity of epithelial cells.
In accordance with this aspect of the invention there are provided novel polypeptides of human origin referred to herein as cytostatin II as well as biologically, diagnostically or therapeutically useful fragments, variants, homologs, analogs, and derivatives thereof.
Among the particularly preferred embodiments of this aspect of the invention are variants of human cytostatin II encoded by naturally occurring alleles of the human cytostatin II gene.
It is another object of the invention to provide a process for producing the aforementioned polypeptides, polypeptide fragments variants, analogs, derivatives and fragments thereof.
In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned cytostatin II polypeptides comprising culturing host cells having expressibly incorporated therein an exogenously-derived human cytostatin II-encoding polynucleotide under conditions for expression of human cytostatin II in the host and then recovering the expressed polypeptide.
It is another object of the invention to provide products, compositions, processes and methods for utilizing the aforementioned polypeptides and polynucleotides for biological, clinical and therapeutic purposes, inter alia.
In accordance with certain preferred embodiments of this aspect of the invention, there are provided methods for, among other things: modulating cell growth in vitro, ex vivo or in vivo; assessing cytostatin II expression in cells by determining protein or mRNA; and assaying genetic variation and aberrations, such as defects, in cytostatin II genes.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided probes that hybridize specifically to human cytostatin II sequences.
In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against cytostatin II polypeptides. In certain particularly preferred embodiments in this regard, the antibodies are highly selective for human cytostatin II.
In accordance with another aspect of the present invention, there are provided cytostatin II agonists, such as those which mimic cytostatin II, bind to cytostatin II receptors and elicit cytostatin II-induced responses. Also among such agonists are those which interact with cytostatin II, or with other modulators or receptors, and thereby potentiate the effects of human cytostatin II.
In accordance with yet another aspect of the present invention, there are provided cytostatin II antagonists, such as those which mimic cytostatin II, bind to cytostatin II receptors but do not elicit cytostatin II-induced responses, and those that bind to or interact with human cytostatin II so as to inhibit its effects.
The agonists and antagonists may be used to mimic, augment or inhibit the action of such polypeptides, for example, and they may be used in the treatment of disorders associated with aberrant growth of cells affected by cytostatins, particularly cytostatin II.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.